Presentation is loading. Please wait.

Presentation is loading. Please wait.

Tissue proteases convert CCL23 into potent monocyte chemoattractants in patients with chronic rhinosinusitis  Julie A. Poposki, MS, Anjeni Keswani, MD,

Published byΙολανθη Αγγελόπουλος Modified over 5 years ago

Similar presentations

Presentation on theme: "Tissue proteases convert CCL23 into potent monocyte chemoattractants in patients with chronic rhinosinusitis  Julie A. Poposki, MS, Anjeni Keswani, MD,"— Presentation transcript:

1 Tissue proteases convert CCL23 into potent monocyte chemoattractants in patients with chronic rhinosinusitis  Julie A. Poposki, MS, Anjeni Keswani, MD, Julie K. Kim, MD, Aiko I. Klingler, PhD, Lydia A. Suh, BS, James Norton, MS, Roderick G. Carter, BS, Anju T. Peters, MD, Kathryn E. Hulse, PhD, Leslie C. Grammer, MD, Bruce K. Tan, MD, David B. Conley, MD, Juan C. Jaen, PhD, Thomas J. Schall, PhD, Robert C. Kern, MD, Atsushi Kato, PhD  Journal of Allergy and Clinical Immunology  Volume 137, Issue 4, Pages e9 (April 2016) DOI: /j.jaci Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Posttranslational modification by NP trypsin-like serine proteases controls CCL23 activity. A-E, Recombinant CCL23 was incubated with 1 mg/mL NP extracts, 1 mg/mL BSA (control), supernatants from activated mast cells (MC), neutrophils (Neut), eosinophils (Eos), M1 macrophages (M1) and M2 macrophages (M2), tryptase βII, or skin chymase for 6 (Fig 1, A and C-E) or 0 to 24 (Fig 1, B) hours. CCL23 was incubated with NP extract in the presence of DMSO, PIC, aprotinin (Apr), nafamostat mesylate (Naf), leupeptin (Leu), E-64, pepstatin A (PepA), K579, marimastat (Mar), UK (UK), SSR69071 (SSR), TLCK, or tosyl phenylalanyl chloromethyl ketone (TPCK) for 6 hours (Fig 1, C). CCL23, cleaved CCL23, and CCL23 (46-120) were detected by means of Western blotting with an anti-CCL23 antibody. Results are representative of 3 separate experiments with separate donors (Fig 1, B-D). F, THP-1 cells were incubated with NP-treated CCL23 (solid circles), the same concentration of NP extracts without CCL23 (solid squares), or BSA-treated CCL23 (solid triangles) for 2 hours by using a microchemotaxis plate (n = 4). RFU, Relative fluorescence units. *P < .05, 1-way ANOVA, compared with medium control (Fig 1, F). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 CCL23 (47-117) is an active CCL23 metabolite in NP extracts. NP extract–treated CCL23 proteins were separated by using SDS-PAGE. A, N-terminal protein sequences of each product were detected by using an Edman-based sequencer. B, CCL23 was incubated with NP extracts for 1 to 6 hours. Truncated products were detected by using MALDI-TOF mass spectrometry. C, Summary table of identified truncated products assigned by comparison of measured with predicted m/z values by using MALDI-TOF mass spectrometry and N-terminal sequencing. D, Protein sequence of CCL23. Arrows indicate identified cleavage sites. Red color indicates the sequence of the potential major active metabolite CCL23 (47-117) in NPs. Results are representative of 3 separate experiments with separate donors (Fig 2, A-C). E, THP-1 cells were incubated with CCL23, CCL23 (46-120), or CCL23 (47-117) for 2 hours by using a microchemotaxis plate (n = 4). F, THP-1 cells were incubated with 1 nmol/L CCL23 (47-117) for 2 hours by using a microchemotaxis plate in the presence of 0.02% DMSO (vehicle control) or 100 nmol/L CCX8960 (n = 3). RFU, Relative fluorescence units. *P < .05, 1-way ANOVA, compared with medium control (Fig 2, E). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 Trypsin-like serine protease inhibitor blocks truncation of CCL23 by NP extracts and tryptase. Recombinant CCL23 (2 μg/mL) was preincubated with 1 mg/mL NP extracts (A) or 15 μg/mL recombinant tryptase βII (B) in the presence or absence of 200 μmol/L TLCK or 200 μmol/L TPCK for 3 hours. CCL23 and cleaved CCL23 were detected by means of Western blotting with an anti-CCL23 antibody. Results are representative of 3 separate experiments. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 NP-treated CCL23 has potent chemoattractant activity for PBMCs. CCL23 (50 μg/mL) was preincubated with 1 mg/mL NP extracts for 6 hours. The migration assay was performed in a 96-well microplate format FluoroBlok microchemotaxis system (3-μm pore, BD Biosciences). PBMCs were labeled with Calcein AM dye and resuspended in phenol red–free RPMI-1640 containing 0.1% BSA. Labeled PBMCs were incubated with 200 ng/mL (A) or 10 to 200 ng/mL (B) CCL23 (blue symbols), NP-treated CCL23 (red symbols), the same concentration of NP extracts without CCL23 (open symbols), or medium alone (black symbols) for 0.5 to 4 (Fig E2, A) or 2 (Fig E2, B) hours by using a microchemotaxis plate (n = 5). Migrated cells were analyzed with a SpectraMAX Gemini EM microplate reader (excitation at 494 nm and emission at 517 nm). *P < .05, 1-way ANOVA, compared with medium control. RFU, Relative fluorescence units. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 NP-pretreated CCL23 has potent chemoattractant activity for THP-1 cells through CCR1. A, Recombinant CCL23 (5 μmol/L) was preincubated with 1 mg/mL NP extracts for 0 to 24 hours, and then THP-1 cells were incubated with 1 nmol/L NP-treated CCL23, the same concentration of NP extracts without CCL23, and 1 nmol/L untreated CCL23 for 2 hours by using a microchemotaxis plate (n = 5). B, THP-1 cells were incubated with 1 nmol/L NP-treated CCL23 and 1 nmol/L CCL2 for 2 hours by using a microchemotaxis plate in the presence of 0.02% DMSO (vehicle control) or 100 nmol/L CCX8960 (a CCR1-specific antagonist, n = 3). *P < .05, 1-way ANOVA, compared with medium control. RFU, Relative fluorescence units. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E4 Serine protease inhibitor blocks truncation of CCL23 by NP extracts. CCL23 was incubated with NP extract in the presence of 1% DMSO (vehicle control) or 10 μmol/L nafamostat for 6 hours. Truncated products were detected by using MALDI-TOF mass spectrometry. The x-axis of the mass spectra represents m/z. NP indicates proteins contained in the NP extracts. Numbered peaks refer to corresponding amino acid residues of CCL23. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E5 Synthesis of recombinant CCL23 (47-117). Recombinant CCL23 (47-117) was synthesized at BioLegend. Purity of recombinant CCL23 (47-117) was greater than 98% analyzed by using SDS-PAGE (A) and HPLC (not shown). B, Molecular mass was detected as by using ESI-TOF mass spectrometry. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E6 CCR1 is expressed on macrophages in NPs. We identified the population of macrophage subsets in NPs with the following steps. We first selected singlets (by using forward scatter [FSC]-A/FSC-W and side scatter [SSC]-A/SSC-W), excluded dead cells (Aqua+), selected the CD45+ population, excluded granulocytes (SSChigh), and then selected CD68+ macrophages. A, Representative flow cytometric plots for M1 macrophages (CD163−CD206−) and M2 macrophages (CD163+CD206+, n = 3). B, Expression of CCR1 was determined on M1 and M2 macrophages. FMO, Fluorescence minus one. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E7 CCR1 is expressed on mDCs in NPs. We identified the population of DC subsets in NPs with the following steps. We first selected singlets (by using forward scatter [FSC]-A/FSC-W and side scatter [SSC]-A/SSC-W), excluded dead cells (Aqua+), selected the CD45+ population, and then removed granulocytes (SSChigh) and B cells (CD19+). We then identified DC subsets by the following markers: mDC1 (CD1c+CD11c+), mDC2 (CD141highCD11c+), and plasmacytoid dendritic cells (pDC; CD303+). CCR1 expression was determined on mDC1s, mDC2s, pDCs, and CD19+ B cells (negative control). FMO, Fluorescence minus one. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Download ppt "Tissue proteases convert CCL23 into potent monocyte chemoattractants in patients with chronic rhinosinusitis  Julie A. Poposki, MS, Anjeni Keswani, MD,"

Similar presentations

Proton pump inhibitors decrease eotaxin-3/CCL26 expression in patients with chronic rhinosinusitis with nasal polyps: Possible role of the nongastric.

Proton pump inhibitors decrease eotaxin-3/CCL26 expression in patients with chronic rhinosinusitis with nasal polyps: Possible role of the nongastric.
Anju T. Peters, MD, Atsushi Kato, PhD, Ning Zhang, PhD, David B

Anju T. Peters, MD, Atsushi Kato, PhD, Ning Zhang, PhD, David B
Douglas A. Plager, PhD, Ellen A. Weiss, Gail M. Kephart, BS, Robert M

Douglas A. Plager, PhD, Ellen A. Weiss, Gail M. Kephart, BS, Robert M
Surface availability of beta-glucans is critical determinant of host immune response to Cladosporium cladosporioides  Rachael A. Mintz-Cole, PhD, Eric.

Surface availability of beta-glucans is critical determinant of host immune response to Cladosporium cladosporioides  Rachael A. Mintz-Cole, PhD, Eric.
Flow cytometry imaging identifies rare TH2 cells expressing thymic stromal lymphopoietin receptor in a “proallergic” milieu  Amanda J. Reefer, MS, Kathryn.

Flow cytometry imaging identifies rare TH2 cells expressing thymic stromal lymphopoietin receptor in a “proallergic” milieu  Amanda J. Reefer, MS, Kathryn.
The importance of being “pure” neutrophils

The importance of being “pure” neutrophils
Margaretha A. Faber, MD, Vito Sabato, MD, Chris H

Margaretha A. Faber, MD, Vito Sabato, MD, Chris H
Proton pump inhibitors decrease eotaxin-3/CCL26 expression in patients with chronic rhinosinusitis with nasal polyps: Possible role of the nongastric.

Proton pump inhibitors decrease eotaxin-3/CCL26 expression in patients with chronic rhinosinusitis with nasal polyps: Possible role of the nongastric.
Douglas A. Plager, PhD, Ellen A. Weiss, Gail M. Kephart, BS, Robert M

Douglas A. Plager, PhD, Ellen A. Weiss, Gail M. Kephart, BS, Robert M
Heterogeneous inflammatory patterns in chronic rhinosinusitis without nasal polyps in Chicago, Illinois  Bruce K. Tan, MD, MS, Aiko I. Klingler, PhD,

Heterogeneous inflammatory patterns in chronic rhinosinusitis without nasal polyps in Chicago, Illinois  Bruce K. Tan, MD, MS, Aiko I. Klingler, PhD,
Proprotein convertases generate a highly functional heterodimeric form of thymic stromal lymphopoietin in humans  Julie A. Poposki, MS, Aiko I. Klingler,

Proprotein convertases generate a highly functional heterodimeric form of thymic stromal lymphopoietin in humans  Julie A. Poposki, MS, Aiko I. Klingler,
Evidence for intranasal antinuclear autoantibodies in patients with chronic rhinosinusitis with nasal polyps  Bruce K. Tan, MD, Quan-Zhen Li, PhD, Lydia.

Evidence for intranasal antinuclear autoantibodies in patients with chronic rhinosinusitis with nasal polyps  Bruce K. Tan, MD, Quan-Zhen Li, PhD, Lydia.
Reduced IFN-γ receptor expression and attenuated IFN-γ response by dendritic cells in patients with atopic dermatitis  Eva Gros, MSc, Susanne Petzold,

Reduced IFN-γ receptor expression and attenuated IFN-γ response by dendritic cells in patients with atopic dermatitis  Eva Gros, MSc, Susanne Petzold,
Anju T. Peters, MD, Atsushi Kato, PhD, Ning Zhang, PhD, David B

Anju T. Peters, MD, Atsushi Kato, PhD, Ning Zhang, PhD, David B
Factor XII–independent cleavage of high-molecular-weight kininogen by prekallikrein and inhibition by C1 inhibitor  Kusumam Joseph, PhD, Baby G. Tholanikunnel,

Factor XII–independent cleavage of high-molecular-weight kininogen by prekallikrein and inhibition by C1 inhibitor  Kusumam Joseph, PhD, Baby G. Tholanikunnel,
Peanut oral immunotherapy results in increased antigen-induced regulatory T-cell function and hypomethylation of forkhead box protein 3 (FOXP3)  Aleena.

Peanut oral immunotherapy results in increased antigen-induced regulatory T-cell function and hypomethylation of forkhead box protein 3 (FOXP3)  Aleena.
Abnormal CD161+ immune cells and retinoic acid receptor–related orphan receptor γt– mediate enhanced IL-17F expression in the setting of genetic hypertension 

Abnormal CD161+ immune cells and retinoic acid receptor–related orphan receptor γt– mediate enhanced IL-17F expression in the setting of genetic hypertension 
Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis  Kaori Mukai, PhD, Nicolas Gaudenzio,

Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis  Kaori Mukai, PhD, Nicolas Gaudenzio,
Identification of a distinct glucocorticosteroid-insensitive pulmonary macrophage phenotype in patients with chronic obstructive pulmonary disease  Kirandeep.

Identification of a distinct glucocorticosteroid-insensitive pulmonary macrophage phenotype in patients with chronic obstructive pulmonary disease  Kirandeep.
Type 3 innate lymphoid cells induce proliferation of CD94+ natural killer cells  Shuo Li, PhD, Hideaki Morita, MD, PhD, Beate Rückert, Sci Tec, Tadech.

Type 3 innate lymphoid cells induce proliferation of CD94+ natural killer cells  Shuo Li, PhD, Hideaki Morita, MD, PhD, Beate Rückert, Sci Tec, Tadech.

Similar presentations

Ads by Google